Incubation of photolyzed bovine rod outer segment (ROS) membranes with pertussis toxin led to an inhibition of GTPase activity in an NAD-dependent reaction; the decrease in GTPase activity paralleled an increase in [32-P]ADP-ribosylation of a 39k Da protein which is the Alpha-subunit of transducin (TAlpha). ADP-ribosylation of TAlpha occurred in both photolyzed and dark ROS membranes. Neither ATP nor Gpp(NH)p was required for ADP-ribosylation or inhibition of GTPase. Incubation of ROS with choleragen or pertussin toxin prevented subsequent [32-P]ADP-ribosylation by the same but not the other toxin; i.e., the two toxins label different sites on TAlpha. TAlpha, which is [32-P]ADP-ribosylated by pertussin toxin in purified transducin, was also labeled by the toxin after separation from TBetaGamma (36k Da and about 10k Da); neither component of TBetaGamma was a pertussis toxin substrate. Labeling of TAlpha was enhanced by TBetagamma (maximally at a molar ratio of about 1:1). Limited proteolysis by trypsin of TAlpha in the presence of Gpp(NHH)p produced sequentially proteins of 38 and 32K Da. The amino terminus of both 38 and 32K Da proteins was leucine; that of a TAlpha was apparently blocked. The 32K Da peptide was not a pertussin toxin substrate. Poor labeling of the 38K Da peptide was not a pertussis toxin substrate. Poor labeling of the 38K Da protein was poor and was not enhanced by TBetaGamma. Trypsin treatment of [32-P]ADP-ribosyl-TAlpha produced a labeled 37-38K Da doublet followed by appearance of 32-P at the dye front. Without rhodopsin, labeling of TAlpha (in the presence of TBetaGamma) was unaffected by Gpp(NH)p, GTPGammaS, GTP, GDP, or GDPBetaS but was increased by ATP. With photolyzed rhodopsin and TBetaGamma, Gpp(NH)p and GTPBetaS decreased labeling. Pertussis toxin ADP-ribosylates the inhibitory guanine nucleotide-binding subunit of adenylate cyclase (Gi) but not the stimulatory subunit (Gs); choleragen ADP-ribosylated Gs but not Gi. Since both toxins ADP-ribosylate TAlpha, it appears that this protein may possess characteristics of both Gs and Gi.